B. megaterium strain QM B 1551 harbors a multiplasmid array of seven plasmids, 11% of the total cellular DNA. The long-term goal of this study is to thoroughly investigate one multiplasmid "genome" to provide much needed information on plasmid biology, the role of plasmids in the cell, plasmid transfer and distribution among the Gram-positive bacteria, and the potential role in the emergence of new pathogens. Three of the smallest plasmids (5.4, 9.1 and 26kb) have been completely sequenced and a fourth (55 kb) is 70% sequenced. Six replicons have been characterized, and genes potentially of use in bioremediation, transfer and vector construction, as well as a complete rRNA operon have been found. This proposal is a continuation of a grant in which the replicon of the third largest plasmid, pBM500 (71 kb) has been characterized, flanking DNA cloned, the source plasmid identified, and a study initiated to test host range. A pilot study of a pathogenic B. megaterium isolate (the first ever reported) has led to the discovery that genes cross-hybridizing with B. cereus virulence genes are present on the chromosome of the isolate and strain QM B 1551. This proposal is for a considerable expansion of the first grant in which the remaining plasmid DNA (400 kb) will be shotgun-cloned and sequenced in collaboration with Integrated Genomics. Annotation and initial functional genomics will be done using powerful new programs at both NIU and IG. This is the first attempt to completely sequence and characterize a multiplasmid array and should reveal many interesting plasmid genes for further annotation and functional analysis. From results with the smaller plasmids, it is expected that genes for transfer, unusual metabolic enzymes, and other unknown but significant processes will be found. To continue testing for host range and horizontal transfer, the replicons will be used in hybridizations, conjugations, and transformations to other Bacillus and other Gram positive genera to determine the host range, distribution, and transfer capabilities of the plasmids. The information obtained should substantially increase the database of plasmid genes and lead to a more comprehensive understanding of the role of these plasmids in the cell and of their potential applications in medicine, bioremediation and vector construction.